Faculty Bibliography

In the United States, laboratories frequently offer multiple different assays for testing of cerebrospinal fluid (CSF) samples to provide laboratory support for the diagnosis of central nervous system Lyme disease (CNSLD). Often included among these diagnostic tests are the same enzyme immunoassays and immunoblots that are routinely used to detect the presence of antibodies to Borrelia burgdorferi in serum. However, performing these assays on CSF alone may yield positive results simply from passive diffusion of serum antibodies into the CSF. In addition, such tests are only United States Food and Drug Administration-cleared and well-validated for testing serum, not CSF. When performed using CSF, positive results from these assays do not establish the presence of intrathecal antibody production to B. burgdorferi and therefore should not be offered. The preferred test to detect intrathecal production of antibodies to B. burgdorferi is the antibody index assay, which corrects for passive diffusion of serum antibodies into CSF and requires testing of paired serum and CSF collected at approximately the same time. However, this assay also has limitations and should only be used to establish a diagnosis of CNSLD in conjunction with patient exposure history, clinical presentation and other laboratory findings.

BACKGROUND:Health care and public health professionals rely on accurate, real-time monitoring of infectious diseases for outbreak preparedness and response. Early detection of outbreaks is improved by systems that are comprehensive and specific with respect to the pathogen but are rapid in reporting the data. It has proven difficult to implement these requirements on a large scale while maintaining patient privacy. OBJECTIVE:The aim of this study was to demonstrate the automated export, aggregation, and analysis of infectious disease diagnostic test results from clinical laboratories across the United States in a manner that protects patient confidentiality. We hypothesized that such a system could aid in monitoring the seasonal occurrence of respiratory pathogens and may have advantages with regard to scope and ease of reporting compared with existing surveillance systems. METHODS:We describe a system, BioFire Syndromic Trends, for rapid disease reporting that is syndrome-based but pathogen-specific. Deidentified patient test results from the BioFire FilmArray multiplex molecular diagnostic system are sent directly to a cloud database. Summaries of these data are displayed in near real time on the Syndromic Trends public website. We studied this dataset for the prevalence, seasonality, and coinfections of the 20 respiratory pathogens detected in over 362,000 patient samples acquired as a standard-of-care testing over the last 4 years from 20 clinical laboratories in the United States. RESULTS:The majority of pathogens show influenza-like seasonality, rhinovirus has fall and spring peaks, and adenovirus and the bacterial pathogens show constant detection over the year. The dataset can also be considered in an ecological framework; the viruses and bacteria detected by this test are parasites of a host (the human patient). Interestingly, the rate of pathogen codetections, on average 7.94% (28,741/362,101), matches predictions based on the relative abundance of organisms present. CONCLUSIONS:Syndromic Trends preserves patient privacy by removing or obfuscating patient identifiers while still collecting much useful information about the bacterial and viral pathogens that they harbor. Test results are uploaded to the database within a few hours of completion compared with delays of up to 10 days for other diagnostic-based reporting systems. This work shows that the barriers to establishing epidemiology systems are no longer scientific and technical but rather administrative, involving questions of patient privacy and data ownership. We have demonstrated here that these barriers can be overcome. This first look at the resulting data stream suggests that Syndromic Trends will be able to provide high-resolution analysis of circulating respiratory pathogens and may aid in the detection of new outbreaks.

The extended-spectrum beta-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae, we identified three clinical isolates, CN1, CR14 and NY9, carrying both blaCTX-M and blaKPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, blaCTX-M and blaKPC were carried on two different plasmids. In contrast, CN1 had one blaKPC-2 and three copies of blaCTX-M-15 integrated in the chromosome, for which the blaCTX-M-15 genes were linked to an insertion sequence ISEcp1, whereas blaKPC-2 gene was in the context of a Tn4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn4401a-blaKPC-2-prophage genomic island, CN1 also carried a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of Type I-E CRISPR-cas in K. pneumoniae strains; and suggested that the evolving CRISPR-cas with its acquired novel spacer might have induced the mobilization of antimicrobial resistance genes from plasmids into chromosome. The integration and dissemination of multiple copies of blaCTX-M and blaKPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae Additionally, the implications from this study also raise concern for the application of a CRISPR-cas strategy against antimicrobial resistance.

Introduction: Klebsiella pneumoniae belongs to the ESKAPE group and is one of the top five pathogens causing nosocomial infections worldwide. The widespread emergence of multidrug-resistant K. pneumoniae is a major public health concerns. beta-lactams are the antibiotics most widely used all over the world, have also given rise to a continuous increase of resistance and driven diversification of the resistance and driven diversification of the resistance mechanisms.Various beta-lactamases have been identified to hydrolyze beta-lactams. The extended-spectrum beta-lactamases, such as CTX-M and SHV, hydrolyze cephalosporins and monobactams; whereas K. pneumoniae carbapenemases (KPCs) confer carbapenem resistance. Many beta-lactamase genes reside in plasmids, transferring between different stains or species. Methods: Routine clinical MicroScan and Etest methods were used to profile the susceptibility of antimicrobial resistance in K. pneumoniae clinical isolates recovered from patient specimens during 2005-2014. Additional PCR amplification followed by Sanger sequencing was used for gene detection of blaCTX-M and blaKPC. A combination of short- and long-read whole genome sequencing was used for three isolates positive to both blaCTXM and blaKPC. Comparative genomic analyses were further conducted to characterize detailed resistance gene structures and genomic features. Results: We identified three K. pneumoniae isolates, CN1, CR14 and NY9, carrying both blaCTXM and blaKPC. Through de novo assembly, we obtained three complete genomes. In CR14 and NY9, blaCTX-M and blaKPC were carried in distinctive plasmids with distinctive incompatibility. In contrast, CN1 harbors in chromosome one blaKPC-2 and three blaCTX-M-15 genes, in addition to one blaSHV-11. Whereas blaCTX-M-15 genes were transferred via the same insertion sequence ISEcp1, blaKPC-2 gene was mobilized in the context of insertion sequence Tn4401a, which was found conjugative with a PsP3-like prophage in CN1 chromosome. Intriguingly, downstream of the newly generated Tn4401a-prophage genomic island, CN1 carried a CRISPR-Cas array with four spacers targeting a variety of K. pneumoniae plasmids carrying antimicrobial resistance genes. It suggests the evolving CRISPR-Cas, a bacterial immune system preventing phage infection and plasmid transfer, might have induced the mobilization of resistance genes from plasmids into chromosome under the selective force. Conclusion: We observed the emergence of K. pneumoniae isolates carrying multiple choromosomal beta-lactamase genes. Our comparative genomic analyses revealed their shared genomic features and dynamic evolutionary events occurring in choromosome.The dissemination of beta-lactam resistance from plasmids to chromosome depicts the currently complex pandemic scenario of multidrug resistant K. pneumoniae isolates

The diagnosis of Lyme disease is a controversial topic. Most practitioners and scientists recognize that Lyme disease is associated with certain objective clinical manifestations supported by laboratory evidence of infection with Borrelia burgdorferi sensu lato (the etiologic agent). There are others, however, who believe that patients with Lyme disease may have a wide variety of entirely nonspecific symptoms without any objective clinical manifestation and that laboratory evidence of infection by B. burgdorferi is not required to support the diagnosis. In reality, this perspective is not evidence based and would inevitably lead to innumerable misdiagnoses, given the high frequency of medically unexplained symptoms, such as fatigue and musculoskeletal pains, in the general population. Although those espousing this viewpoint do not believe that a positive laboratory test is required, nevertheless, they often seek out and promote alternative, unapproved testing methods that frequently provide false-positive results to justify their diagnosis. Herein, we provide a brief overview of Lyme disease testing, emphasizing current usage and limitations. We also discuss the use of nonvalidated procedures and the prospects for a reduction in such testing practices in the future.